This proposal investigates the first hours of anthrax infections; in vivo germination of the Bacillus anthracis endospore, macrophage survival, growth and escape of the vegetative bacilli. Outside the host, endospores remain metabolically-dormant, preserving virulence even when exposed to harsh environmental conditions. Endospores are the anthrax contagion, entering the body where they are phagocytosed by regional macrophages. Endospores "sense" the new locale, germinate and outgrow to a vegetative state. Our preliminary data defined discrete mutants blocked at each of these steps. After escape, massive bactermia, toxemia and death ensues. Our data also indicate that anthrax endospores have unique in vivo sensory and signaling mechanisms for triggering germination. Germination occurs rapidly in cultured macrophages. Non-pathogenic Bacillus sp. endospores show no increased germination in macrophages. A B. anthracis transposon-mediated mutagenesis system allowed selection of individual endospore mutants incapable of germination in macrophages but fully capable of germination and outgrowth in bacterial media. Several unique classes of mutants were characterized. One such loci, named gerP (germination Plasmid), is located on the virulence (toxin) plasmid pXO1. Thus mutations in gerP eliminate host-specific germination but not general germination responses. The aims of this proposal are to: a. define and characterize the germination genes of B. anthracis and host chemical signals to determine their roles in the host- specific germination response; b. determine defined intracellular events and bacterial genes used by the vegetative bacilli allowing for survival and escape from the macrophage, and; c. understand the relevance of B. anthracis host-specific germination systems and early intracellular events in terms of pathogenesis in the murine model. Knowledge of these critical "establishment" stages of anthrax may provide targets for early intervention after exposure to anthrax endospores. Understanding this rapid and dramatic switch, from absolute metabolic dormancy of the endospore to growing virulent bacilli allows anthrax to be exploited as an effectual model for examining the earliest stages of bacterial infectious cycles.